Genetic assays and strains using human TP53

ABSTRACT

Yeast strains carrying a human wild-type TP53 are employed to select for mutations. The types of mutations can be analyzed genetically as recessive or dominant-negative. The mutational spectrum of dominant-negative TP53 mutants selected in yeast correlates tightly with TP53 mutations found in human cancers. Thus the use of such yeast assays is validated for studying the effects of various agents on human TP53, one of the most important and ubiquitous of human cancer genes. Assays, kits, and constructs are provide which use yeast as a genetic system for making and studying human TP53 mutations. Such assays can be used to develop therapeutic agents, to study putative carcinogens, and to identify other cellular components which interact with p53 and abrogate its activity.

This application is a divisional of U.S. Ser. No. 08/650,125, filed May1, 1996, now U.S. Pat. No. 5,830,751.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grantsawarded by the National Institutes of Health.

BACKGROUND OF THE INVENTION

More than half of all human cancers are associated with one or morealterations in the tumor suppressor gene TP53 (1-4). Many premalignantlesions, a subset of malignant clones, and germlines of families proneto cancer are characterized by the presence of one wild-type and onemutant allele of TP53 (5-9). In this situation the mutant p53 proteinmay act in a dominant-negative fashion, ultimately leading to loss ofheterozygosity and thus a further growth advantage for the malignantcells. Alternatively, the mutant p53 protein may have acquired a newtumor promoting activity which is independent of wild-type p53. Thesehypotheses are based on the analysis of only a few TP53 mutationsusually in the setting of over-expression of the mutant protein, andtheir relevance to TP53 mutations in general has not been proven (8,10-13).

There is a need in the art for additional systems in which to studymutations in human p53, an important and ubiquitous cancer suppressorgene.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a yeast cell useful forselecting and studying mutations in human p53.

It is another object of the invention to provide a method of identifyingcompensatory mutations in TP53 which suppress dominant-negative TP53mutant phenotypes.

It is still another object of the invention to provide a method ofidentifying potential therapeutic agents.

It is yet another object of the invention to provide a method ofscreening putative carcinogens.

It is yet another object of the invention to provide a method foridentifying cellular proteins which interact with p53 and abrogate itsactivity.

It is an object of the invention to provide a kit for isolatingmutations in p53.

It is another object of the invention to provide a gene fusion usefulfor isolating and studying p53 mutations.

These and other objects of the invention are provided by one or more ofthe embodiments described below. In one embodiment of the invention ayeast cell is provided. The cell comprises a first reporter gene whichis selectable or counterselectable. The reporter gene is operably linkedto a DNA sequence to which human p53 specifically binds. The cell alsocomprises a first fusion gene which expresses a human p53 in the cell.The fusion gene comprises a promoter operably linked to a human p53coding sequence.

In another embodiment of the invention a method of identifyingcompensatory mutations in TP53 which suppress dominant-negative TP53mutant phenotypes is provided. The method involves providing a cellwhich comprises:

(i) a reporter gene which is selectable, wherein the reporter gene isoperably linked to a DNA sequence to which human p53 specifically binds;and

(ii) a first fusion gene which expresses a dominant-negative allele ofhuman p53 in the cell, the fusion gene comprising a promoter operablylinked to a human p53 coding sequence.

Then a population of DNA molecules comprising a second fusion gene isintroduced into the cell. The second fusion gene comprises a promoteroperably linked to a mutagenized human p53 coding sequence. Phenotypicrevertants of the dominant-negative allele of human TP53 are selectedusing the selectable phenotype of the reporter gene.

According to another embodiment of the invention a method of identifyingpotential therapeutic agents is provided. A cell is provided whichcomprises:

a reporter gene which is selectable, wherein the reporter gene isoperably linked to a DNA sequence to which human p53 specifically binds;and

a fusion gene which expresses a dominant-negative allele of human TP53in the cell, the fusion gene comprising a promoter operably linked to ahuman p53 coding sequence.

Test compounds are contacted with the cell. The selectable phenotype ofthe reporter gene is assayed. Desirable test compounds are identified aspotential therapeutic agents if they induce the cell to display theselectable phenotype.

In another aspect of the invention a method of screening putativecarcinogens for their effect on a p53 allele is provided. A cell isprovided which comprises:

a reporter gene which is counterselectable, wherein the reporter gene isoperably lied to a DNA sequence to which human p53 specifically binds;and

a fusion gene which expresses human p53 in the cell, the fusion genecomprising a promoter operably linked to a human p53 coding sequence.

The cell is contacted with a putative carcinogen. Cells are isolatedwhich contain a mutation in the human p53 coding sequence bycounterselecting for loss of expression of the reporter gene.

According to another embodiment of the invention cellular proteins whichinteract with p53 and abrogate its activity are identified. A populationof cells is provided which comprise:

a reporter gene which is counterselectable, wherein the reporter gene isoperably linked to a DNA sequence to which human p53 specifically binds;and

a fusion gene which expresses human p53 in the cell, the fusion genecomprising a promoter operably linked to a human p53 coding sequence.

A library of human cDNA molecules is introduced into the population ofcells. Each of the cDNA molecules is operably linked to expressioncontrol sequences so that the human cDNA is expressed in the cell. Thecells are assayed to identify those which express the counterselectablephenotype of the reporter gene. The counterselectable phenotypeidentifies cells which express a protein which abrogates p53 activity.

According to another aspect of the invention a kit is provided. The kitcomprises three yeast strains. The first yeast strain comprises acentromeric plasmid which itself comprises: a fusion of a yeast alcoholdehydrogenase promoter operably linked to a human p53 coding sequence;and a yeast histidine (HIS3) gene. The first yeast strain also comprisesan integrated reporter gene which consists of a p53 consensus bindingsequence inserted upstream from the URA3 locus. The second yeast straincomprises an integrated reporter gene which consists of a p53 consensusbinding sequence inserted upstream from the URA3 locus. The third yeaststrain comprises a centromeric plasmid which itself comprises a fusionof a yeast alcohol dehydrogenase promoter operably linked to a human p53coding sequence, and a yeast LEU2 gene. The third yeast strain alsocontains an integrated reporter gene which consists of a p53 consensusbinding sequence inserted upstream from the URA3 locus. The first strainis of a compatible mating type to the second and third strains.

In still another embodiment of the invention a tripartite gene fusion isprovided. The fusion comprises a human p53-specific DNA-binding site; ayeast URA3 gene; and a portion of a yeast SPO13 gene. The humanp53-specific DNA-binding site is upstream of the URA3 gene, and theportion of the yeast SPO13 gene is interposed between the URA3 gene andthe human p53-specific DNA-binding site. Moreover, the portion of theyeast SPO13 gene consists of the first 15 codons of SPO13 andnucleotides 5′ to nucleotide −170.

These and other embodiments of the invention provide the art with toolsfor studying mutagenesis and carcinogenesis in general, as well as forstudying the important cancer-related gene TP53.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Phenotypes of p53 mutants selected in yeast. In contrast towild-type p53 (phenotype Ura⁺Foa^(S)) all dominant-negative as well asrecessive mutants are Ura⁻Foa^(R). Upon mating to strains with one ortwo wild-type ADH-p53 expression vectors the dominant-negative mutantscan be classified by their degree of dominance over wild-type p53. Thestronger class 1 interferes with one and two copies of wild-type ADH-p53and thus survives on Foa plates. The weaker class 2 is only dominantover one wild-type copy. For the p53 mutants in bold letters NcoI/StuIfragments with the mutations were recloned into the wild-type ADH-p53plasmid pRB16. The mutants with ** represent hotspot codons which werenot identified by our screen (1, 2, 4) (see Table 3). #2022 and #2026are recessive mutations leading to the expression of truncated p53proteins. The media used were SC−Ura (−U), SC−His+Foa (−H+F),SC−His−Leu+Foa (−H−L+F), and SC−His−Leu−Trp+Foa (−H−L−T+F). The −U and−H+F media test for p53 function (wild-type grows on −U and fails togrow on −H+F). The −H−L+F medium tests for the ability of mutant p53 tointerfere with the function of a single wild-type copy of p53 (presenton a LEU2 plasmid); dominant-negative mutants will grow on this medium.The −H−L−T+F medium tests for the ability of mutant p53 to interferewith the function of two wild-type copies of p53 (present on LEU2 andTRP1 plasmids).

FIG. 2 Comparison of the dominant-negative ADH-p53 mutations selected inyeast to the five hotspot regions of human cancer mutations and toreported germline mutations (Li-Fraumeni syndrome and others). The boxedyeast mutations hit the hotspot regions (2, 5). For codons with shadedbackground germline mutations have been reported (7, 27, 28). The figureshows the clustering of the strongest dominant mutations to codons 179,241-248 and 277-281. Mutations of class 1 are in bold and of class 2 inplain text.

FIG. 3 Western Blot analysis with PAb 1801 (34) for p53 proteinexpression in yeast strains with wild-type and mutant ADH-p53 expressionvectors. Protein levels for the dominant-negative mutants are similar tothat of wild-type p53. The yeast strain with 2 expression vectors forwild-type ADH-p53 shows approximately two-fold more p53 protein than allother strains indicating that the strongest dominant p53 mutants ofclass 1 can in fact override higher levels of wild-type protein. For thep53 mutants in bold letters NcoI/StuI fragments with the mutations werecloned into wild-type ADH-p53. The mutants with ** represent hotspotcodons which were not identified by our screen (1, 2, 4).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

We developed a yeast assay for p53 and its consensus DNA binding site toscreen for and analyze spontaneous dominant-negative p53 mutations. Wehave discovered that such mutations cluster in the mutational hotspotsof human cancers. We have demonstrated different degrees of dominance,the most dominant mutations localizing to codons 179, 241-248 and277-281. These results are fully consistent with a dominant-negativemode of action for the large majority of tumorigenic TP53 alleles.

Our p53 assay is based on the principles of yeast systems designed byFields and others, which allow the study of protein/protein interactionsby simple phenotypic readouts (19-23). The use of a counterselectablemarker, such as URA3, as the reporter gene, allows screening for andagainst p53 expression. In a preferred embodiment, activation of URA3leads to survival on medium lacking uracil, but prevents growth onplates containing 5-fluoro-orotic acid (Foa) due to the conversion ofFoa to a toxic product (resulting in a Ura⁺ Foa^(S)phenotype) (24). Inour assays, URA3 activation depends upon the site-specific binding ofp53 to its consensus DNA binding site (25) placed upstream of URA3; p53expression is driven by the ADH1 promoter from a CEN (centromeric)plasmid (ADH-p53), which is maintained at approximately one copy percell.

Yeast cells are employed for the assays described here, although othercell types, both prokaryotic and eukaryotic, preferably with welldefined genetics, can be used as well. Yeast are particularly usefulbecause of their welleveloped genetic system. Reporter genes usefulaccording to the present invention are selectable or counterselectable,or both. The URA3 gene is particularly useful. Other usefulcounterselectable genes include LYS2, LYS5, CAN1, MET2, MET15, and GAL1.The reporter gene is operably lint to a DNA sequence to which human p53specifically binds. An actual human p53-binding sequence or any sequencewhich conforms to the consensus sequence taught by el-Deiry (25) can beused. Among this family of sequences there may be a slight variation inthe behavior in the assays. As shown by the actual p53-binding humansequences, slight variations from the consensus sequence can be madewhile still enabling p53 to specifically bind. Operable linkage,according to the present invention, means that the DNA sequence isupstream of the reporter gene and close enough so that p53 bindingactivates transcription of the reporter gene. Typically this is withinabout 1 kb.

It is desirable that at least one of the reporter gene constructs of thepresent invention be integrated in the genome. This can be accomplishedreadily by amplification of the construct and introduction into yeast byany technique known in the art. The transformed construct can integratevia homologous recombination at the site of the endogenous reporter geneor at any marker gene site. This event can be readily selected for ifthe genomic target site is mutated and the transforming marker gene iswild-type.

A fusion gene which expresses a human p53 in a yeast cell typically hasp53 coding sequences linked to an endogenous yeast cell promoter. Usefulpromoters are those for “housekeeping” genes which are typicallyexpressed throughout the life cycle at high levels. One such usefulpromoter is that for the alcohol dehydrogenase gene, but others may beused as is convenient. Promoters are operably linked to a TP53 gene whenthey drive transcription of the gene Such promoters are usually upstreamand within about 1 kb of the start of translation.

Suitable TP53 alleles for use in the various embodiments of the presentinvention are wild-type alleles as well as mutants. The mutant allelesmay be those which are experimentally made, but more interesting allelesare those which are found in human cancers. Most interesting alleles arethose which are dominant-negative, which means that the alleles cause amutant phenotype even in the presence of a wild-type allele.

After generation of mutations in TP53 on a first expression plasmid itis often desirable to test the mutant allele in the presence ofwild-type TP53. This can be accomplished by mating with a compatiblehaploid yeast strain which contains a wild-type TP53 expressionconstruct. Preferably the two TP53 expression constructs will producep53 at similar levels, or if not, at levels which can be manipulated,such as by controlled induction. The phenotype of the diploid cellcontaining a mutant and wild-type allele of TP53 can be assayed orobserved, to determine whether the mutation is recessive or dominant.

In another aspect of the invention a second reporter gene is introducedinto the yeast cell which contains a reporter gene as well as a fusiongene expressing p53. The second reporter gene can be selectable,counterselectable, or both. The reporter gene can be the same ordifferent from the first reporter gene. Like the first reporter gene,the second reporter gene is operably linked to a DNA sequence to whichhuman p53 specifically binds. This reporter gene allows one to determinewhether a mutation occurred on the TP53-plasmid or on the genomicreporter gene.

The set of yeast strains and plasmids disclosed here lend themselves toa variety of methods. In one method, mutations which are able tocompensate for a dominant-negative TP53 mutation are identified. Suchcompensatory mutations are of intrinsic scientific interest, todetermine interactions between portions of the p53 multimer. Inaddition, a compensatory mutation which functions in trans to suppress adominant-negative TP53 mutant phenotype will be useful in therapeuticapplications. Such mutations will also be instructive in the design ofdrugs which have the same effect on dominant-negative p53 mutants. Byusing the strains described herein, one can, for example mutagenizehuman p53 coding sequences and introduce them to strains which contain aselectable reporter gene operably linked to a DNA sequence to whichhuman p53 specifically binds, and a TP53 expression construct which hasa dominant-negative mutation. Desirably the mutation will be onecommonly found in human tumors. Using the selectable phenotype of thereporter gene, one can select for a change in phenotype due to theintroduction of a particular mutagenized human p53 coding sequence.

According to another aspect of the invention, potential therapeuticagents can be identified. Using a cell according to the presentinvention which has a selectable reporter gene operably linked to a DNAsequence to which human p53 specifically binds and a fusion gene whichexpresses a dominant-negative allele of human TP53, one can perform adrug screening assay. Preferably the allele is one found in humantumors. Test compounds can be contacted with such cells and one canselect for the ability of the cells to express the reporter gene.Candidate therapeutic agents are able to induce the cell to display theselectable phenotype of the reporter gene.

Another method for which the strains of the present invention areparticularly suited is a screening assay for putative carcinogens. TheSalmonella/mammalian-microsome mutagenicity test developed by Bruce Amesand co-workers has confirmed many carcinogens as mutagens and hasidentified numerous new mutagens. The test is based on a prokaryoticsystem with reporter genes which play no role in human carcinogenesis. Aeukaryotic system which tests the mutagenicity of compounds on, forexample, tumor suppressor genes aids in the classification of and riskassessment for mutagens/carcinogens.

For the tumor suppressor gene p53 there are several examples of mutagenswhich cause specific mutations in the p53 sequence. Theses include,amongst others, UV light leading to skin cancer and aflatoxin B1 leadingto hepatomas. The causal relationships could only be demonstratedbecause of strong epidemiological data for exposure to a single or fewmutagens and a high incidence of the cancer. Similar causalrelationships may exist for other mutagens and cancers; however, theyare more difficult to prove because there is concomitant exposure tovarious mutagens and a lower incidence of the specific cancer. Aeukaryotic mutagenicity assay for the p53 gene enables theidentification of mutagens that cause a specific pattern of p53mutations.

Using a strain which has a counterselectable reporter gene operablylinked to a DNA sequence to which human p53 specifically binds and afusion gene which expresses human p53 in the cell, one can test putativecarcinogens by contacting them with the cell. Using thecounterselection, one can identify carcinogens which have inducedmutations in TP53. If desired the particular mutations can beidentified, for example, by sequencing. Thus particular mutationalfingerprints on TP53 of the carcinogens can be identified. Suchfingerprints can be used epidemiologically, for example to assess theeffects of particular carcinogens on populations.

If desired, one can incubate putative carcinogens with a mammalian liverlysate prior to the incubation with the tester cells. Such liver lysatesare able to metabolize pre-carcinogens to carcinogens, as occurs in thehuman or animal body.

The assay can be performed, for example, using a yeast strain which ishomozygous diploid for the reporter gene UAS53::URA3 and which containsa single copy of wild-type p53 on a centromeric plasmid. Known mutagens,such as UV light, aflatoxin B1 and benzo[α]pyrene, or unknown mutagensare applied to the yeast cells. Mutations which cause a non-functionalp53 protein lead to a phenotype change from Ura⁺Foa^(S) to Ura⁻Foa^(R)which can be easily screened for. Based on our experience with thesystem, other mutations can also lead to Foa resistance, most of whichare recessive mutations in the UAS53::URA3 reporter gene. By using astrain which is homozygous diploid for the reporter gene the frequencyof these mutations will be reduced to 1×10⁻⁶ and should therefore benegligible. In addition, a second wild-type p53 expression construct canbe integrated into the genome. Thus the assay will preferentiallyidentify dominant-negative p53 mutations. The plasmids with the mutatedp53 genes can be recovered from yeast, expressed in E. coli, purified,and sequenced.

The yeast assay for carcinogenic compounds can be performed in thefollowing way:

1. Homozygous diploid UAS53::URA3 yeast cells which are His⁻ and whichbear an episomal p53 expression plasmid with a HIS3 marker gene areplated as a lawn onto −His plates.

2. Crystals, impregnated filter paper disks, or droplets of thecompound(s) to be tested are applied to the lawn, setting up a gradientof the compound as the test cells grow. If the test compound has strongmutagenic potential, p53 mutants arise at higher frequencies in thezones containing higher concentrations of the compound.

3. The lawn of cells is transferred, by replica-plating, to a −His platecontaining 5-Foa, selecting for the p53 plasmid. Only p53 mutants shouldgrow on these plates. Mutagenic effect on p53 is observed as a halo ofFoa-resistant colonies surrounding the spot at which the compounds wereoriginally placed.

4. The plasmids bearing p53 mutations are recovered from the yeastcolonies. The nature of the mutation(s) can be determined by DNAsequencing.

Note that step 1 can be performed, as in the Ames test, by the inclusionof a rat liver homogenate on the plate, providing the enzymes (mixedfunction oxygenases, cytochrome P-450s, etc.) that can activate certainp carcinogens to carcinogens. Furthermore, yeast strains with mutationsin important DNA repair enzymes can be used in order to increase thesensitivity of the test.

Because our system is yeast-based there are two ways in which p53mutagenesis may be different from mutagenesis in human cells. i) Thereis no DNA methylation in yeast. Thus 25 mutagens which affect methylatedbases will not be active in the assay. This can be overcome (ifnecessary) by expression of the appropriate methyltransferase(s) inyeast. ii) p53 cDNA lacks introns, thus mutations at splice junctionswill not be represented. These are known, however, to be very rareevents based on the sequence information of p53 mutations in humancancers. Additionally, some compounds may not be able to enter the yeastcells because of their cell walls. This can be circumvented by thepreparation and use of spheroplasts or by the utilization of yeaststrains with mutations which affect cell permeability.

The strains of the present invention also lend themselves to methods foridentification of other cellular components which interact with p53,either stimulating or inhibiting its binding to its specific bindingsequences. Using a counterselectable reporter gene and a TP53 expressionconstruct, one can introduce a library of human cDNA molecules each ofwhich is operably linked to expression control sequences. Selectingagainst the reporter gene using a counterselective agent, e.g., 5-FOA inthe case of URA3, identifies individual clones which contain a humancDNA which inhibits p53 activity, possibly by means of the protein whichthe cDNA encodes.

This method was tested in a pilot study. A reporter strain(UAS53:URA3/p53) was transformed with exon 2 of the SV40 large T antigen(TAg), a viral antigen known to prevent DNA binding and transactivationby p53. The large T antigen plasmid pGAD-T2 was originally designed fora two-hybrid screen and encodes a Gal4 transactivation domain-TAgfusion. The large T antigen fusion protein was able to interfere withp53 activity in our system, changing the phenotype from Foa^(S) toFoa^(R), while the control vector plasmid pGAD-2F encoding the Gal4transactivation domain alone was unable to do so.

Kits are also contemplated as part of the invention which compriseuseful sets of yeast strains and/or plasmids. In one contemplated kit,three strains are provided. A first yeast strain contains a centromericplasmid. The plasmid contains a gene fusion of a yeast alcoholdehydrogenase promoter operably linked to a human p53 coding sequence;and a yeast histidine (HIS3) gene. The first yeast strain also containsan integrated reporter gene which consists of a p53 consensus bindingsequence inserted upstream from the URA3 locus. This strain can be usedfor selection of TP53 mutants. The other two strains can be used forgenetic characterization of the TP53 mutants which are isolated. Thesecond yeast strain contains an integrated reporter gene which consistsof a p53 consensus binding sequence inserted upstream from the URA3locus, within a distance short enough so that binding of p53 activatesURA3 transcription. The third yeast strain contains a centromericplasmid which contains a fusion of a yeast alcohol dehydrogenasepromoter operably linked to a human p53 coding sequence, and a yeastLEU2 gene. The third yeast strain also contains an integrated reportergene which consists of a p53 consensus binding sequence insertedupstream from the URA3 locus. The first strain is of a compatible matingtype to the second and third strains, such that the first strain can bemated with each of the others to form diploid cells. Examples of each ofthese strains are provided in Table 1. Uses of these strains arediscussed throughout. Written instructions for performing any of theassays described herein may also be enclosed in the kit, as well asmedia and selective agents. Strains are typically packaged separatelyand then bundled or held together in a common container.

Also provided by the present invention is a tripartite gene fusionsuitable for integration into the yeast genome. The gene fusion containsa human p53-specific DNA-binding site, a yeast URA3 gene, and a portionof a yeast SPO13 gene. The human p53-specific DNA-binding site isupstream of the URA3 gene, and the portion of the yeast SPO13 gene isinterposed between the URA3 gene and the human p53-specific DNA-bindingsite. The portion of the yeast SPO13 gene consists of the first 15codons of SPO13 and nucleotides upstream thereof, until nucleotide −170.Importantly, the SPO13 upstream region contains a URS (upstreamrepressing sequence) that prevents basal low level transcription ofURA3. Such a construct is disclosed in detail below, and can be used information of strains useful for the practice of the disclosed methods.

The following examples are provided for exemplification purposes onlyand are not intended to limit the scope of the invention.

EXAMPLES Example 1

Yeast Strains, Plasmids and Isolation of TP53 Mutants

All of the yeast media used here (e.g. −His) were dropout media based onsynthetic complete supplemented minimal medium (14) lacking theindicated nutrient(s). The yeast strains and plasmids used are describedin Table 1.

TABLE 1 Yeast strains Strain Relevant genotype* Plasmids (markers)†RBy33 MATα 1cUAS53::URA3 — RBy41 MATα 1cUAS53::URA3 pRB16 (ADH-p53 HIS3CEA) RBy159 MATα 1cUAS53::URA3 — RBy160 MATα 1cUAS53::URA3 pLS76(ADH-p53 LEU2 CEN) RBy161 MATα 1cUAS53::URA3 pLS76 (ADH-p53 LEU2 CEN)pRB17 (ADH-p53 TRP1 CEA) RBy162 MATα ura3-52 pLS76 (ADH-p53 LEU2 CEN)*All strains listed (except RBy162) are also lys2Δ202 trp1Δ63 his3Δ200leu2Δ1. RBy162 is also lys2Δ202 trp1Δ63 his3Δ200 leu2Δ1 ade2Δ. †pRB16and pRB17 were derived from pLS76 (15) by subcloning the XhoI-SacIfragment containing ADH-p53 (including the CYC1 transcriptionterminator) into CEN vectors pRS413 and pRS414 (16), respectively.

Construction of Reporter Gene

We fused the SPO13 promoter to a sequence encoding a fusion protein withthe first 15 amino acids of SPO13 and the URA3 protein (SPO13::URA3 inpPL128). The SPO13::URA3 fragment was excised from pPL128 and clonedinto a pBSK plasmid (Stratagene). The resulting plasmid, pMV252,contains EcoRI sites at −170 and −368 in the SPO13 promoter.

For construction of the UAS53::URA3 reporter genes, oligonucleotidescorresponding to the p53 consensus DNA binding site(JB820:5′-AATTTAGGCATGTCTAGGCATGTCTA-3′ (SEQ ID NO:1) andJB821::5′-AATTTAGACATGCCTAGACATGCCTA-3′ (SEQ ID NO:2) (14) wereannealed, phosphorylated, and ligated into EcoRI-digested pMV252.

The UAS53::URA3 alleles were integrated at the ura3-52 locus byhomologous recombination of the product of a PCR reaction. The 5′ primerused was JB516 that contains 40 nucleotides of the URA3 sequenceupstream of its promoter (−257 to −218) fused to 20 nucleotides of theSPO13 promoter (−370 to −351(11):5′-GAAGGTTAATGTGGCTGTGGTTTTCAGGGTCCATAAAGCTTGTCCTGGAAGTCTCATGGAG-3′ (SEQ ID NO:3). The 3′ primer used was 3′URA3 (URA3 sequence+656 to +632 (12)):5′-TCAGGATCCCTAGGTTCCTTTGTTACTTCTTCCG-3′ (SEQ IDNO:4) .

Isolation of Independent TP53 Mutations

For isolation of independent TP53 mutations, patches of single coloniesfrom RBy41 (containing an ADH-p53 HIS3 expression vector (pRB16) and theintegrated reporter gene 1cUAS53::URA3) were grown on synthetic completemedium without histidine (SC−His plates), replica-plated to SC−His+0.15%5-fluoro-orotic acid (Foa) plates and incubated for 2 to 4 days at 37°C. until 5-fluoro-orotic acid resistant (Foa^(R)) papillae emerged.

Only one single Foa^(R) colony was isolated from each parental patch.These Foa^(R) clones were 1) mated to RBy159 (MATα, isogenic to RBy41,but lacking an ADH-p53 expression vector) and replica-plated to SC−Uraplates and 2) mated to RBy160 (RBy159 with the ADH-p53 LEU2 plasmidpLS76 (15)) followed by replica-plating to SC−His−Leu plates to selectfor diploids and then SC−His−Leu+0.15% Foa plates to evaluate thedominance/recessivity of the Foa^(R) phenotype. Clones which were Ura⁺in mating assay #1 and Foa^(S) in assay #2 were recessive and were notdue to TP53 plasmid-dependent mutations. Most of these clones representrecessive mutations that knock out 1cUAS53::URA3. Clones which were Ura⁻in assay #1 and Foa^(S) in assay #2 were TP53 plasmid-dependentrecessive mutations. Only clones which were Foa^(R) in assay #2potentially contained a dominant-negative TP53 plasmid-dependentmutation; these were further characterized by growing themnon-selectively and isolating strains which had lost the (potentiallymutated) pRB16. A wild-type TP53 expression plasmid was then introducedinto these strains as follows. The plasmid-free strains were mated toRBy162 (MATα ura3-52 and containing pLS76), replica-plated to SC−Ade−Leuplates to select for diploids, followed by replica-plating of thediploids to SC−Leu+0.15% Foa plates. Foa^(R) clones which regained theirFoa^(S) phenotype as a result of these manipulations were judged tocontain dominant-negative TP53 plasmid-dependent mutations.

Example 2

Identification and Classification of Dominant-negative TP53 Mutants

We isolated a total of 49 independent spontaneous p53 mutants thatbehaved in a dominant-negative fashion. These mutants were identifiedusing a two-step selection procedure. In the first step, haploid yeastcolonies deficient in URA3 expression were selected on plates containingFoa. In the second step, these colonies were mated to strains containingeither the wild-type reporter gene or one copy of wild-type ADH-p53 andsubsequently transferred to plates containing Foa. Dominant-negativealleles of TP53 showed an Foa^(R) phenotype in both cases. Recessivealleles of TP53 or cis-acting reporter-linked mutations exhibited anFoa^(S) phenotype in the presence of an additional copy of wild-typeADH-p53 or the wild-type reporter gene, respectively.

Characterization of TP53 mutants. The mutant pRB16 plasmids from allidentified dominant-negative TP53 plasmid dependent clones wererecovered in bacteria (17), retransformed into RBy33 (RBy41 withoutpRB16) and the phenotypes rechecked. The dominant-negative phenotypeswere then further classified by testing the degree of the dominance overone or two doses of wild-type ADH-p53 as follows. The retransformedstrains bearing mutant pRB16 derivatives were mated to RBy160 and RBy161(RBy159 containing two ADH-p53 expression plasmids, pLS76 (15) andpRB17, which is identical to pRB16 except for the selectable marker TRP1(16)), replica-plating them to SC−His−Leu and SC−His−Leu−Trp platesrespectively, replica-plating them to the same selective plates with0.15% Foa and incubating them at 30° C. for 2 to 4 days.

Some of the dominant-negative TP53 mutants were isolated as falsepositives from a cDNA library screen that is irrelevant to thisinvention; these mutants were characterized in the same fashion (Table2). Due to the fact that this subset of the mutants studied did notnecessarily represent independent isolates, a numerical analysis ofmutation frequencies within this subset would be meaningless.

The recessive plasmid-dependent TP53 mutants were also recovered intobacteria and retransformed. These isolates (as well as the dominantisolates) were evaluated by immunoblotting with anti-p53 antibody PAb1801, performed as described. RBy50 (pRS413 (16) in RBy33) was used asthe negative control.

TABLE 2 Properties of independent TP53 mutations selected in yeast Totalnumber of Foa^(R) clones 717 Number of TP53 plasmid-dependent mutants111* Recessive mutants  67 (9%) Dominant mutants  31 (4%)^(†) class 1 13 class 2  18 *13 plasmid-dependent mutants could not be classified,since they did not show consistent phenotypes before and after plasmidrecovery and retransformation. ^(†)18 additional independentdominant-negative mutants were obtained as false positives in a cDNAlibrary screen. These mutants are independent by virtue of a uniquemutation and are identified by * in Table 3.

Recessive mutations in the reporter gene were found in 87% and in theTP53 gene in 9% of all mutants. 4% of the Foa^(R) colonies containeddominant-negative TP53 mutations (Table 2). Once the dominant-negativeTP53 mutants had been identified, the TP53 plasmids were recovered andtransformed into a fresh reporter strain (RBy33) to exclude artifacts ofthe original strain. In all cases the same dominant-negative phenotypecould be reproduced. The dominant-negative mutants could be furtherclassified by mating them to a strain with two wild-type ADH-p53plasmids thus characterizing the dominance of the mutant proteins in thepresence of two doses of the wild-type ADH-p53 gene. The most dominantmutants were able to interfere with one and two copies of wild-typeADH-p53 (class 1). Less dominant TP53 mutants could only override theactivity of a single wild-type allele (class 2) (FIG. 1). These classesrepresented 43% and 57% of the dominant-negative TP53 mutants,respectively.

Example 3

Sequences of the Dominant-negative TP53 Mutants

We then sequenced the core domains (codons 102-292) of the 49dominant-negative mutants.

Miniprep DNA (17) for the plasmids was RNase treated (7 mg/ml, 10 min,37° C.), extracted with phenol/chloroform and sequenced with Taqpolymerase (Perkin-Elmer) using Prizm kit dye-terminator cyclesequencing on an Applied Biosystems 373A Stretch automated sequencer.Sequences were analyzed using Sequencher software (Gene CodesCorporation, Inc., Ann Arbor, Mich.) for the Macintosh. The core domainsof ADH-p53 were sequenced using primers JB990 (5′-ACCAGCAGCTCCTACACC-3′)(SEQ ID NO:5) and JB991 (5′-GAGGAGCTGGTGTTGTTG-3′)(SEQ ID NO:6). Eightdominant-negative clones (bold numbers in Table 3) were further analyzedby ligating NcoI/StuI fragments with the mutations (base pairs 477 to1039) into pRB16 using standard methods (18). Wild-type sequence for theC-terminal parts of these fragments was verified by sequencing withprimers JB1052 (5′-CCATCCTCACCATCATCAC-3′) (SEQ ID NO:7) and JB1091(5′-GCAGGGGAGGGAGAGATGG-3′) (SEQ ID NO:8). The hotspot mutations forcodons 175 and 249 (in Table 3) were cloned into pRB16 using the samestrategy. Phenotypes were checked as described above.

Forty-one of the dominant-negative mutants had a single missensemutation and 8 had an in-frame deletion. Very strikingly, the mutationsclustered around five of the six known hotspot codons in the TP53 gene:245, 248, 249, 273 and 282 (1, 2, 4). We identified 5 mutations in codon245, 2 in 248 and 2 in 273. 88% of the missense mutations hit the fivehotspot regions for mutations (132-143, 151-159, 172-179, 237-249 and272-286) or codons for which germline mutations have been described(FIG. 2) (2, 5, 7, 27, 28). 96% of the mutations we recovered in yeasthave been described in human cancers or cancer cell lines (Table 3) (4,29, 30). Our screen hit 5 of the 7 amino acids important in direct DNAbinding (codons 241, 248, 273, 277 and 280) and 3 of the 4 amino acidsinvolved in zinc atom contact (codons 176, 179 and 242) (31-33).

With the exception of H179N, all of the most dominant mutations(class 1) localized to codons 241-248 and 277-281. 83% of the mutationsin these two regions had the class 1 phenotype (FIG. 2) indicating astrong correlation between the location of mutations and their degree ofdominance.

TABLE 3 Sequence data on dominant-negative p53 mutations selected inyeast described in Mutation Mutation cancer Number Codon NucleotideAmino Acid Class (29, 30)  32* 127 TCC −> CCC Ser −> Pro 2 no  27* 132AAG −> AAC Lys −> Asn 2 yes  26* 135 TGC −> TTC Cys −> Phe 2 yes  43*151 CCC −> CGC Pro −> Arg 2 yes  67 151 CCC −> CAC Pro −> His 2 yes  30*158 CGC −> CCC Arg −> Pro 2 yes  76 176 TGC −> CGC Cys −> Arg 2 yes  17*179 CAT −> AAT His −> Asn 1 yes  50* 236 TAC −> GAC Tyr −> Asp 2 yes  64241 TCC −> TTC Ser −> Phe 1 yes  70 242 TGC −> TTC Cys −> Phe 2 yes  13*244 GGC −> GAC Gly −> Asp 1 yes  14* 244 GGC −> AGC Gly −> Ser 1 yes 12* 245 GGC −> AGC Gly −> Ser 1 yes  16* 245 GGC −> CGC Gly −> Arg 1yes  55 245 GGC −> AGC Gly −> Ser 1 yes  57 245 GGC −> AGC Gly −> Ser 1yes 101* 245 GGC −> GAC Gly −> Asp 1 yes  41* 246 ATG −> ATT Met −> Ile2 yes  62 246 ATG −> AGG Met −> Arg 1 yes  1* 248 CGG −> TGG Arg −> Trp1 yes  63 248 CGG −> TGG Arg −> Trp 1 yes  48* 252 CTC −> ATC Leu −> Ile2 no  65 252 CTC −> ATC Leu −> Ile 2 no  20* 257 CTG −> CCG Leu −> Pro 2yes  37* 257 CTG −> CAG Leu −> Gln 2 yes  36* 259 GAC −> TAC Asp −> Tyr2 yes  29* 265 CTG −> CCG Leu −> Pro 2 yes  69 273 CGT −> CCT Arg −> Pro2 yes  74 273 CGT −> CCT Arg −> Pro 2 yes  7* 277 TGT −> TAT Cys −> Tyr1 yes  28* 278 CCT −> CAT Pro −> His 2 yes  38* 278 CCT −> TCT Pro −>Ser 2 yes  10* 279 GGG −> GAG Gly −> Glu 1 yes  53 279 GGG −> GAG Gly −>Glu 1 yes  61 279 GGG −> GAG Gly −> Glu 1 yes  8* 280 AGA −> ACA Arg −>Thr 1 yes  58 280 AGA −> AGC Arg −> Ser 1 no  3* 281 GAC −> GGC Asp −>Gly 1 yes  5* 281 GAC −> TAC Asp −> Tyr 1 yes  56 281 GAC −> GGC Asp −>Gly 1 yes  18*, 68, Δ175-180 (or 176-181 or 177-182^(†) 2 yes  68,  71, 72,  73,  75  35* Δ216 (or 217 or 218)^(‡) 2 yes  42* Δ252-254 (or251-253)^(§) 175^(¶) CGC −> CAC Arg −> His 2 yes 249^(¶) AGG −> AGT Arg−> Ser 1 yes Bold clones were characterized further by cloning themutation into wild-type ADH-p53 and rechecking the phenotypes. *Thesedominant-negative mutations were obtained as false positives in a cDNAlibrary screen. ^(†)This deletion presumably arises frequently becauseof the direct repeat GCGCTGC present at codons 175-176 and 181-182.^(‡)deletion of one of three tandem GTG codons. ^(§)Direct repeat of ATCflanks deleted nucleotides. ^(¶)These hot spot mutations were clonedinto wild-type ADH-p53 since our screen did not identify mutations ofthese codons.

To exclude that second mutations up- or downstream of the core domaincontributed to the described phenotypes, we subcloned NcoI/StuIfragments (codons 159-347 encoding only the mutation of interest asconfirmed by sequencing) into a wild-type ADH-p53 plasmid for thefollowing mutants: C176R, D175-180, D217, G245D, R248W, R273P, P278S andD281 Y. In all cases the same dominant-negative phenotype was reproduced(FIG. 1, Table3).

Our screen hit 3 hotspot amino acids (codons 245, 248 and 273) butfailed to identify mutations in the other 3 (codons 175, 249 and 282).These hotspots in human cancers are due in large part to methylation ofthe CpG dinucleotides present in codons 175 and 282 and exposure toaflatoxin B₁ for codon 249 (1-4, 8); neither situation applies to ouryeast system. Two amino acid substitutions for these hotspots, R175H andR249S, were subdloned into wild-type ADH-p53 and shown to preventUAS53::URA3 transcription. These mutants were also found to be dominantover wild-type (FIG. 1, Table 3).

Example 4

Protein Expression Levels of Dominant-negative TP53 Mutants

The wild-type and the mutant ADH-p53 genes are expressed from the samepromoter in our system. In order to investigate whether thedominant-negative phenotypes were partially caused by an increasedstability of the mutant protein we analyzed protein levels byimmunoblotting with anti-p53 antibody PAb 1801 (34). FIG. 3 shows thatprotein levels for the mutant p53 proteins were similar to that ofwild-type.

Example 5

Analysis of Recessive TP53 Mutants

We also analyzed the more abundant recessive TP53 mutants. Since weconsidered the likelihood of non-missense mutations high, weimmunoblotted protein extracts from the 67 independently obtainedrecessive TP53 mutants. None of these clones showed full-length protein.Four mutants expressed shorter proteins consistent with C-terminaltruncation since PAb 1801 recognizes the N-terminus (34).

Conclusions

We have used the methods and strains described here to isolate andanalyze TP53 mutations. Based on our work in yeast, where recessive TP53mutations outnumbered dominant ones by about two to one, we believe thatrecessive TP53 mutations probably occur at a higher rate in human cellsthan dominant mutations, but that the recessive mutations are much lesslikely to lead to cancer (and therefore to be sequenced) since theremaining wild-type allele continues to exert its important functions.Our selection in yeast for dominant-negative TP53 mutations hasidentified a variety of missense mutations and in-frame deletions whoselocations show a striking correlation with the hotspot regions of humancancer mutations. This suggests that the high frequency of human cancermutations in these hotspot regions is in large part due to theirdominant-negative effect on the wild-type p53 protein. Our data showsthat the dominant negative mutants interfere with the wild-type proteinto varying degrees, thus the amount of residual p53 activity in cellsheterozygous for different TP53 mutations is likely to be different.However, even for the strongest dominant-negative mutants, there islikely to be some residual p53 function. The dominant-negativeinterference with the function of wild-type p53 should lead to elevatedrates of DNA damage, chromosome loss, and other forms of loss ofheterozygosity of the TP53 locus. Loss of heterozygosity would eliminatethe residual activity provided by the wild-type TP53 allele and providethe (pre-)malignant clone with further growth advantages.

Class 1 p53 mutants in our assay are more proficient than class 2mutants in interfering with wild-type p53 function. The locations of allclass 1 mutations correspond closely to areas of the core domain whichare essential for the structure of the DNA binding surface of p53 (L2loop, codons 163-195 and L3 loop, codons 236-251), for major groovecontacts in the pentamer sequence of the consensus DNA binding site (H2a helix of the loop-sheet-helix motif, codons 278-286) and for minorgroove contacts in the A T-rich region of the binding site (L3 loop)(31-33). These mutations may be more efficient in destabilizing aheterotetramer of mutant and wild-type p53. Assuming i) a single mutantsubunit can poison a p53 tetramer, ii) equal size pools of mutant andwild-type protein and iii) unbiased mixing of mutant and wild-typesubunits, heterozygous dominant mutations should lower p53 activity16-fold. Thus, overexpression of a dominant-negative mutant relative towild-type is theoretically not required for abrogation of wild-type p53function, and our experiments in yeast confirm this. These data suggestthat the mutant p53 overexpression observed in human cancers representsan additional level of complexity in p53 deregulation.

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein, however, is notto be construed as limited to the particular forms disclosed, since theyare to be regarded as illustrative rather than restrictive. Variationsand changes may be made by those skilled in the art without departingfrom the spirit of the invention.

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8 26 base pairs nucleic acid single linear 1 AATTTAGGCA TGTCTAGGCATGTCTA 26 26 base pairs nucleic acid single linear 2 AATTTAGACATGCCTAGACA TGCCTA 26 60 base pairs nucleic acid single linear 3GAAGGTTAAT GTGGCTGTGG TTTCAGGGTC CATAAAGCTT GTCCTGGAAG TCTCATGGAG 60 34base pairs nucleic acid single linear 4 TCAGGATCCC TAGGTTCCTT TGTTACTTCTTCCG 34 18 base pairs nucleic acid single linear 5 ACCAGCAGCT CCTACACC18 18 base pairs nucleic acid single linear 6 GAGGAGCTGG TGTTGTTG 18 19base pairs nucleic acid single linear 7 CCATCCTCAC CATCATCAC 19 19 basepairs nucleic acid single linear cDNA 8 GCAGGGGAGG GAGAGATGG 19

What is claimed is:
 1. A method of identifying compensatory mutations inTP53 which suppress dominant-negative TP53 mutant phenotypes comprisingthe steps of: providing a cell comprising: (i) a reporter gene which isselectable, wherein the reporter gene is operably linked to a DNAsequence to which human p53 specifically binds; and (ii) a first fusiongene which expresses a dominant-negative allele of human p53 in thecell, the fusion gene comprising a promoter operably linked to a humanp53 coding sequence; introducing into the cell a population of DNAmolecules comprising a second fusion gene, wherein the second fusiongene comprises a promoter operably linked to a mutagenized human p53coding sequence; and selecting for phenotypic revertants of thedominant-negative allele of human TP53 using the selectable phenotype ofthe reporter gene, wherein a phenotypic revertant which contains saidsecond fusion gene identifies said second fusion gene as comprising acompensatory mutation in TP53 which suppresses dominant-negative TP53mutant phenotypes.
 2. The method of claim 1 wherein thedominant-negative allele of human TP53 is an allele found in a humantumor.
 3. The method of claim 1 wherein the cell is a yeast cell.